Heterogeneity of extracellular vesicles in porcine myoblasts regulates adipocyte differentiation

Figures

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  Fig. 1. Skeletal muscle stem cell-derived small extracellular vesicles inhibit porcine adipose precursor cell viability. (a) Transmission electron microscopy of sEV derived from porcine skeletal muscle stem cells demonstrated (Note: Blue arrows indicate small extracellular vesicles, scale bar = 200 nm). (b) Nanoparticle tracking analysis was used to determine the size distribution and concentration of sEV. (c) Western Blot was performed to detect sEV marker proteins Alix, TSG101, CD9, and CD63. (d) sEV-treated porcine primary adipocytes were assayed for cell viability by MTT assay (n = 6). Values are presented as means ± SEM, *P < 0.05, and **P < 0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups.

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  Fig. 2. Skeletal muscle stem cells-derived small extracellular vesicles significantly inhibit adipogenesis. (a) Oil Red O staining of porcine primary adipocytes treated with PBS (CON) or sEV (scale bar = 50 μm). (b) Statistical results of TG content of porcine primary adipocytes treated with PBS or sEV (n = 4). (c–h) Real-time quantitative PCR assay of adipogenesis-related genes PPARγ, C/EBPα, fatty acid synthesis related gene CD36, FABP4, ACC, and pro-inflammatory factor IL-6 in porcine primary adipocytes treated with sEV (n = 6). (i, j) Gray-scale results of PPARγ protein expression by Western Blot after sEV treatment of porcine primary adipocytes (n = 6). Values are presented as means ± SEM, *P < 0.05, and **P < 0.01, according to the non-paired Student’s t-test or one-way ANOVA between individual groups.

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  Fig. 3. Insulin or oleic acid treatment of MuSCs-derived sEV reverses the inhibition of adipogenesis. (a) Oil Red O staining of porcine primary adipocytes treated with PBS (CON), sEV, sEV + INS, and sEV + OA (scale bar = 50 μm). (b) TG contents of differentiated porcine primary adipocytes treated with PBS, sEV, sEV + INS, and sEV + OA (n = 6). (c–h) Real-time quantitative PCR detection of adipogenesis-related genes PPARγ, C/EBPα, and fatty acid synthesis-related gene CD36, FABP4 ACC, and pro-inflammatory factor IL-6 in porcine primary adipocytes treated with PBS, sEV, sEV + INS, and sEV + OA (n = 12). (i, j) Western Blot results of adipogenesis-related gene PPARγ in porcine precursor adipocytes (n = 4). The results were presented as mean ± standard error of the mean (SEM). The letters a, b, and c represent the level of statistical significance of the difference between the groups. Different letters mean a significant difference, and the same letters mean the difference is not significant.

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  Fig. 4. MuSCs-derived sEV miR-146a-5p is a key gene that inhibits adipocyte differentiation. (a) High-throughput screening and comparison of miRNA expression abundance in adipocyte-derived sEV and MuSCs-derived sEV. (b) After treatment with PBS, sEV, sEV + INS, and sEV + OA, differentiated porcine precursor adipocytes were induced to mature, and the expression levels of miR-146a-5p, miR-129a, miR-9–3, and miR-30e were detected by real-time quantitative PCR (n = 6). The results were presented as mean ± standard error of the mean (SEM). The letters a and b, represent the level of statistical significance of the difference between the groups. Different letters mean a significant difference, and the same letters mean the difference is not significant.

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  Fig. 5. miR-146a-5p enriched in MuSCs-derived sEV is a key molecule inhibiting adipocyte differentiation. (a) After treatment of sEV and transfections of INC (control) or miR-146a-5p inhibitor, the maturation of porcine precursor adipocytes was induced, and oil red O staining was performed (scale bar = 50 μm). (b) After sEV were processed and transfected with INC or inhibitor, porcine precursor adipocytes were induced to mature, and TG content was detected (n = 6). (c–g) After sEV were processed and transfected with INC or inhibitor, porcine precursor adipocytes were induced to mature, and the adipogenesis-related genes PPARγ, C/EBPα, and fatty acid synthesis-related gene CD36, FABP4, and pro-inflammatory factor IL-6 was detected by real-time quantitative PCR (n = 6). (h, i) After sEV were processed and transfected with INC or inhibitor, porcine precursor adipocytes were induced to mature, and adipogenesis-related gene PPARγ was detected by Western Blot (n = 6). The letters a and b, represent the level of statistical significance of the difference between the groups. Different letters mean a significant difference, and the same letters mean the difference is not significant.